Antimicrobial and Anti-Inflammatory Effects of Cecropin A(1-8)–Magainin2(1-12) Hybrid Peptide Analog P5 against Malassezia furfur Infection in Human Keratinocytes  Sunhyo Ryu, Soon-Yong Choi, Samudra Acharya, Young-Jin Chun, Catherine Gurley, Yoonkyung Park, Cheryl A. Armstrong, Peter I. Song, Beom-Joon Kim  Journal of Investigative Dermatology  Volume 131, Issue 8, Pages 1677-1683 (August 2011) DOI: 10.1038/jid.2011.112 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cecropin A–magainin 2 (CA–MA) hybrid peptide analog P5 inhibits the expression of IL-8 induced by Malassezia furfur (M. furfur) infection in normal human keratinocytes. (a) Total RNA was obtained from cultured normal human keratinocytes, which were infected by M. furfur (27:1) for 8hours in the presence or absence of 0.8μM P5 or P4. The expression of IL-8 mRNA was measured by real-time RT-PCR using human IL-8-specific sense and antisense primers, as described in “Materials and Methods”. The relative intensity of each mRNA expression was normalized with mRNA expression of 18S rRNA. (b) The secretion of IL-8 induced by the M. furfur infection was measured by ELISA, as described in “Materials and Methods”. Cultured HK cell supernatants were collected 24hours after infection with M. furfur (27:1) in the presence or absence of 0.8μM P5 or P4. HK treatment with 0.8μM P5 or P4 alone served as controls. The data shown are representative of triplicate experiments. All values are expressed as mean±SD. Statistically significant differences in the expression of IL-8 mRNA and the secretion of IL-8 were determined by analysis of variance with probabilities shown for both the overall significance and the pairwise comparison (*P<0.001). Journal of Investigative Dermatology 2011 131, 1677-1683DOI: (10.1038/jid.2011.112) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Malassezia furfur (M. furfur)-induced NF-κB nuclear translocation was inhibited by P5 in normal human keratinocytes. Immunolocalization of NF-κB was determined by immunofluorescent staining of cellular NF-κB using chamber slide-cultured normal human keratinocytes, which were infected by M. furfur (27:1) for 30minutes in the presence or absence of 0.8μM P5 or P4. NF-κB was detected by specific anti-NF-κB p65 polyclonal antibodies (anti-NF-κB pAbs). FITC-labeled NF-κB (green) was shown with Hoechst-stained nucleus (blue). Images were acquired with the QICAM fast 1394 camera (McCrone Microscopes & Accessories, Westmont, IL). (a) Non-treated; (b) treated with M. furfur; (c) treated with M. furfur plus P5; and (d) treated with M. furfur plus P4. Bars=20μm. Journal of Investigative Dermatology 2011 131, 1677-1683DOI: (10.1038/jid.2011.112) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 P5 inhibited Malassezia furfur (M. furfur)-induced intracellular calcium fluctuation in normal human keratinocytes. Normal human keratinocytes were grown on glass coverslip culture dishes to approximately 50–70% confluency. Then 2μM fluorescent calcium probe fura-2/acetylmethyl was incorporated. Intracellular calcium fluctuation was determined, as described in “Materials and Methods”. (a) HK cells were treated with M. furfur at a yeast cell to HK ratio of 27:1. (b) HK cells were preincubated with 0.8μM P5 or P4 for 2hours, then treated with M. furfur (27:1). (c, d) HK cells were treated with 0.8μM P5 (c) and P4 (d) alone. Intracellular free calcium concentration (nM) was determined by measuring the ratio of fluorescence at excitation wavelengths of 340 and 380nm. Each peak in the figure represents the rapid intracellular calcium response of individual cell. Arrows are the points of treatment with M. furfur (a, b), P5 (c), and P4 (d), respectively. Journal of Investigative Dermatology 2011 131, 1677-1683DOI: (10.1038/jid.2011.112) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 P5 inhibits the expression of Toll-like receptor 2 (TLR2) induced by Malassezia furfur (M. furfur) infection in normal human keratinocytes. (a) Total RNA was obtained from cultured normal human keratinocytes, which were infected by M. furfur (27:1) for 8hours in the presence or absence of 0.8μM P5 or P4. The expression of TLR2 mRNA was measured by real-time RT-PCR using human TLR2-specific sense and antisense primers, as described in “Materials and Methods”. The relative intensity of each mRNA expression was normalized with mRNA expression of 18S rRNA. The data shown are representative of triplicate experiments. All values are expressed as mean±SD. Statistically significant differences in the expression of TLR2 mRNA were determined by analysis of variance, with probabilities shown for both the overall significance and the pairwise comparison (*P<0.001). (b) Chamber slide-cultured normal human keratinocytes were infected by M. furfur (27:1) for 24hours in the presence or absence of 0.8μM P5 or P4. Immunolocalization of TLR2 was determined by immunofluorescent staining using specific anti-TLR2 polyclonal antibodies, as described in “Materials and Methods”. FITC-labeled TLR2 (green) was shown with Hoechst-stained nucleus (blue). (i) Non-treated; (ii) treated with M. furfur; (iii) treated with M. furfur plus P5; and (iv) treated with M. furfur plus P4. Bars=20μm. Journal of Investigative Dermatology 2011 131, 1677-1683DOI: (10.1038/jid.2011.112) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions